Project Summary Invariant Natural Killer T (iNKT) cells play a central role in several immune responses in diverse biological contexts ranging from autoimmunity, injury and infections to pregnancy and metabolic disease as well as cancer. iNKT cells are characterized by their use of a very limited T cell antigen receptor (TCR) repertoire to recognize lipid antigens presented by CD1d, a non-polymorphic major histocompatibility complex class I-like antigen-presenting molecule. A number of functionally distinct iNKT cell subpopulations, each with propensity to traffic to different tissues and to secrete different cytokines upon activation, have been defined. Although it is clear that these fate assignments are conferred upon iNKT cells during selection in the thymus, the environmental cues that direct these decisions are unknown. Our preliminary data show that 1) in wildtype mice, the avidity of the iNKT TCR correlates with iNKT cell subsets and the expression of markers reflecting strength of signaling during selection; 2) the development of iNKT cells in mice with a fixed TCR repertoire is affected similarly on different genetic backgrounds; 3) the V? usage and CDR3? sequences are unique to each iNKT cell subset; 4) signaling and gene expression in the earliest definable stage of iNKT cell development, immediately after engagement of the TCR by natural ligands in vivo, is altered in mice with reduced TCR signaling. These mice also exhibit a cell intrinsic defect in development of iNKT cell subsets. Based on these preliminary studies, we hypothesize that iNKT cell differentiation and function is determined by TCR specificity and signal strength. This hypothesis will be tested by pursuing the following specific aims: 1) Determine the basis for strain differences in thymic iNKT subset composition; 2) Determine the effect of affinity and ligand- specificity of the TCR on development of iNKT cell subsets. The proposal is innovative because it directly explores the role of TCR specificity/affinity for self-ligands in specifying the development of iNKT cell subsets. The proposed research is significant because it will inform our understanding of iNKT cell subset development and dynamics, a pre-requisite to immune intervention aimed at manipulating and optimizing iNKT cell-based therapies.